Diagnosis of carbon monoxide (CO) poisoning is done traditionally by detecting carboxyhemoglobin (COHb) level. However, the severity of clinical manifestations is unrelated to the absolute levels of COHb. Netrin-1 (Net-1) is a protein with anti-inflammatory and anti-apoptotic properties while asymmetric dimethylarginine (ADMA) is an oxidative stress indicator. This study aimed at assessing and comparing between Net-1, ADMA and COHb as biomarkers in CO poisoning. This cross-sectional clinical study was carried out on 30 acutely CO poisoned adults admitted to Benha Poisoning Control Unit, Benha University Hospitals; 30 chronic CO exposed workers at four different car service centres, plus 30 healthy controls. Results showed a positive correlation between serum levels of Net-1 and ADMA and the severity of acute CO poisoning both on admission, and after oxygen therapy. There was a positive correlation between COHb level and both serum levels of Net-1 and ADMA in chronic cases. In conclusion: COHb level can only support the initial diagnosis of acute CO poisoning. Both ADMA and Net-1 are better biomarkers in assessing the severity of acute CO poisoning, where ADMA is better than Net-1. In diagnosis of chronic CO poisoning, both ADMA and Net-1 are as dependable as COHb.